Barbara Mlinar

New insights into adipose tissue dysfunction in insulin resistance

Abstract In a state of caloric excess, adipose tissue plays an essential role by storing lipids. Its expandability determines the onset of metabolic syndrome (central obesity, dyslipidemia, glucose intolerance and hypertension). When the adipocyte endoplasmic reticulum is no longer capable of processing the excess nutrients, the so-called “endoplasmic reticulum stress” develops. This triggers efflux of free fatty acids from adipocytes into the circulation and causes triglyceride overload in skeletal muscle, liver and pancreas. Adipose tissue hypoxia then develops, due to the failure of vasculature to expand with adipocyte hypertrophy. Increased catabolism in mitochondria leads there to oxidative stress. Both phenomena cause deranged adipokine secretion and low-grade inflammation. Inflammatory cytokines, reactive oxygen species and ectopic lipid deposition are the main mediators of insulin resistance and vascular impairment, which both lead finally to diabetes type 2 and cardiovascular disease. Recently, fibrosis of adipose tissue was also demonstrated in obesity, contributing to the interplay of deleterious factors forcing inflammation. The present paper reviews recent evidence for adipose tissue dysfunction, trying to define causes and consequences. In conclusion, insulin resistance and associated complications originate from excess lipids, which cannot be stored without limit in adipose tissue, thus affecting its integrity and adipokine secretion.

Impact of metformin and rosiglitazone treatment on glucose transporter 4 mRNA expression in women with polycystic ovary syndrome

OBJECTIVE The insulin-resistant state of the polycystic ovary syndrome (PCOS) was found to be associated with a decreased glucose transporter GLUT4 expression in the insulin target tissues. This study was performed to explore whether the well-known clinical, hormonal and metabolic efficacy of metformin or rosiglitazone treatment is reflected in the modulation of adipocyte GLUT4 mRNA expression in patients with PCOS. METHODS We enrolled 35 women with PCOS. They received either metformin or rosiglitazone for 6 months. A history, blood samples for the measurement of androgens and s.c. adipose tissue samples were taken at baseline and end point. Quantification of GLUT4 mRNA expression in adipose tissue was performed using real-time quantitative PCR. Homeostasis model assessment (HOMA(IR)) score calculation was applied as a measure for insulin resistance (IR). RESULTS GLUT4 mRNA expression in adipose tissue increased significantly in both groups (P<0.001). The increase was more pronounced in the rosiglitazone group (P=0.040). There was a statistically significant improvement of HOMA(IR) in both groups (P=0.008). After treatment, frequencies of menstrual bleeding were significantly higher (P<0.001) and serum total testosterone levels significantly lower in both groups (P=0.001). CONCLUSIONS A 6-month therapy with insulin sensitizers resulted in marked improvement in adipose tissue GLUT4 mRNA expression in PCOS patients, rosiglitazone being more effective when compared with metformin. The augmentation of the insulin signal transduction was accompanied by a significant improvement of HOMA(IR), menstrual pattern and androgen profile.

Mutations in galactose-1-phosphate uridyltransferase gene in patients with idiopathic presenile cataract

Summary: Impaired activity of the enzyme galactose-1-phosphate uridyltransferase (GALT) has been proposed as a risk factor for idiopathic presenile cataract. A study was undertaken to determine the prevalence of the three most common mutations in the GALT gene (Q188R, K285N and N314D, including its variant Duarte-2) in a group of Slovenian patients with idiopathic presenile cataract. GALT activity was determined in the erythrocytes of 30 cataract patients. DNA was isolated from their blood and analysed for Q188R, K285N and N314D mutations and IVS5−24G>A intronic variation by means of polymerase chain reaction and digestion with restriction enzymes. The average GALT activity of the cataract group was 19.5±4.9 U/g Hb, which is lower than the normal range (p = 0.034). Frequencies of Q188R, K285N, N314D and Duarte-2 alleles in the cataract group were 0.00%, 5.0%, 11.7% and 3.3%, respectively. Only the frequency of the K285N mutation was significantly higher in the patient group than in the control group (p = 0.0244). Our results support the reported association of decreased GALT activity with idiopathic presenile cataract. Molecular analysis indicates that, in the Slovenian population, this association is linked to the K285N mutation, which is neonatally benign in heterozygotes.

Decreased lipin 1β expression in visceral adipose tissue is associated with insulin resistance in polycystic ovary syndrome

OBJECTIVE In polycystic ovary syndrome (PCOS), insulin resistance (IR) appears with high prevalence and represents the major cause of cardiometabolic complications. Lipin 1beta regulates lipid metabolism and augments insulin sensitivity. The impact of lipin 1beta expression in visceral and subcutaneous adipose tissue of PCOS patients on IR was studied for the first time. METHODS Eighty-five PCOS patients and 44 controls were enrolled for subcutaneous tissue biopsy, of whom 25 patients and 30 controls also underwent visceral adipose tissue biopsy. Gene expression of lipin 1beta was measured, together with that of peroxisome proliferator-activated receptor gamma, lipoprotein lipase, hormone-sensitive lipase, adiponectin and glucose transporter 4 in subcutaneous and visceral adipose tissue. Markers of obesity, IR and PCOS were also measured. RESULTS In PCOS patients, lipin 1beta expression in both adipose depots was lower than in controls: 0.76 (0.67-0.84) vs 1.16 (0.90-1.43) for visceral and 0.91 (0.73-1.10) vs 1.30 (1.03-1.57) for s.c. depot (both P<10(-4)). The difference remained significant after adjustment for body mass index (BMI) and also when comparing only lean patients with lean controls. In PCOS patients, visceral adipose lipin 1beta expression correlated negatively with homeostasis model assessment-IR (r=-0.474, P=0.017), BMI (r=-0.511, P=0.009) and waist circumference (r=-0.473, P=0.017), waist circumference remaining significant (P=0.027) in multiple regression. Subcutaneous lipin 1beta expression in PCOS correlated negatively with BMI, waist circumference and plasma triglycerides, and positively with high density lipoprotein-cholesterol. Subcutaneous, but not visceral lipin 1beta expression, correlated positively with the studied genes. CONCLUSIONS Lipin 1beta appears to be involved in the pathogenesis of IR in PCOS.

Association between 11β-hydroxysteroid dehydrogenase type 1 gene polymorphisms and metabolic syndrome

Introduction: The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the conversion of the hormonally inactive cortisone to active cortisol, thus facilitating glucocorticoid receptor activation in target tissues. Increased expression of 11β-HSD1 in adipose tissue has been associated with obesity and insulin resistance. In this study, we investigated the association of two 11β-HSD1 gene (HSD11B1) polymorphisms with the metabolic syndrome (MetS) and its characteristics in the Bosnian population. Materials and methods: The study included 86 participants: 43 patients diagnosed with MetS and 43 healthy controls. Subjects were genotyped for two HSD11B1 gene polymorphisms: rs846910: G>A and rs45487298: insA, by the high resolution melting curve analysis. Genotype distribution and an influence of genotypes on clinical and biochemical parameters were assessed. Results: There was no significant difference in the mutated allele frequencies for the two HSD11B1 gene polymorphisms between MetS patients and controls. In MetS patients, no significant associations between disease-associated traits and rs45487298: insA were found. Regarding rs846910: G>A variant, heterozygous patients (G/A) had significantly lower systolic (P = 0.017) and diastolic blood pressure (P = 0.015), lower HOMA-IR index (P = 0.011) and higher LDL-cholesterol levels (P = 0.049), compared to the wild-type homozygotes. In the control group, rs45487298: insA polymorphism was associated with lower fasting plasma insulin levels (P = 0.041), lower homeostasis model assessment insulin resistance (HOMA-IR) index (P = 0.041) and lower diastolic blood pressure (P = 0.048). Significant differences between rs846910: G>A genotypes in controls were not detected. Haplotype analysis confirmed the association of rs45487298: insA with markers of insulin resistance in the control subjects. Conclusions: Our results indicate that a common rs45487298: insA polymorphism in HSD11B1 gene may have a protective effect against insulin resistance.

Expression of 11β-hydroxysteroid dehydrogenase type 1 in visceral and subcutaneous adipose tissues of patients with polycystic ovary syndrome is associated with adiposity.

Polycystic ovary syndrome (PCOS) is characterized by insulin resistance (IR) and central obesity. The impact of adipose tissue cortisol reactivation by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) on markers of obesity and IR was assessed in PCOS patients. Eighty-five PCOS patients and 43 controls were enrolled for subcutaneous adipose tissue biopsy; 25/85 patients and 29/43 controls underwent also visceral adipose tissue biopsy. HSD11B1 gene expression and expression of lipid metabolism genes were measured in subcutaneous and visceral adipose tissues. Anthropometric and biochemical markers of IR and PCOS were also assessed. HSD11B1 expression in visceral and subcutaneous adipose tissue was increased in PCOS patients compared to controls (p<0.05). After BMI adjustment, the difference was no longer significant. In PCOS patients, visceral HSD11B1 expression correlated positively with waist circumference (p=0.001), BMI (p=0.002), plasma insulin (p<0.05), systolic blood pressure (p=0.003), and lipoprotein lipase (LPL), hormone-sensitive lipase (LIPE) and peroxisome-proliferator activated receptor γ gene expression. Subcutaneous HSD11B1 expression correlated positively with BMI, waist circumference (p<0.001 for both) and HOMA-IR (p=0.003), and negatively with LPL, LIPE, adiponectin and glucose transporter GLUT4 gene expression. HSD11B1 expression in both depots showed a negative correlation with plasma HDL-cholesterol (p<0.03) and a positive one with C-reactive protein (p<0.001). In multiple regression analysis, HSD11B1 expression in visceral adipose tissue was most prominently associated with waist circumference, and that in subcutaneous adipose tissue with BMI (p<0.001 for both). Our results show that PCOS is not associated with increased HSD11B1 expression once adiposity is controlled for. Increased expression of this gene correlates with markers of adiposity and predicts IR and an unfavorable metabolic profile, independently of PCOS.

Frequencies of Q188R and N314D mutations and IVS5-24g>A intron variation in the galactose-1-phosphate uridyl transferase (GALT) gene in the Slovenian population.

Abstract Numerous mutations in the galactose-1-phosphate uridyl transferase (GALT) gene have been found to impair GALT activity to different extent, causing galactosemia. This disorder exhibits considerable allelic heterogeneity in different populations and ethnic groups. The Q188R mutation accounts for 60–70% of classical galactosemia alleles in the Caucasian population. Individuals homoallelic for Q188R have a severe phenotype with complete loss of enzyme activity. Another form of GALT deficiency is Duarte galactosemia with N314D mutation associated alleles (Duarte-2). Although heterozygotes for classical galactosemia are asymptomatic at birth and Duarte galactosemia appears to be quite benign, there are some indications that these disorders can increase the risk of developing certain diseases later in life. The aim of our study was to analyze a healthy Slovenian population for the frequencies of Q188R and N314D mutations, and for the Duarte-2 indicative intronic variation IVS5-24G>A. DNA samples from 174 healthy subjects were analyzed for all three mutations by polymerase chain reaction and digestion with restriction enzymes. Allele frequencies for Q188R and N314D mutations and IVS5-24G>A intron variation were found to be 0.29%, 8.0% and 5.7%, respectively. These results correlate well with those reported for most other healthy Caucasian populations.

Association of LPIN1 gene variations with markers of metabolic syndrome in population from Bosnia and Herzegovina.

AIM To investigate association of two LPIN1 gene variations with main traits of metabolic syndrome (MS) (waist circumference, body mass index, blood pressure, triglycerides, HDL-cholesterol and fasting glucose levels) in population from Bosnia and Herzegovina. METHODS This study included 43 patients with metabolic syndrome and 43 healthy controls from General Hospital in Tešanj, Bosnia and Herzegovina. Subjects were genotyped for two LPIN1 gene variations (rs11693809: C>T and rs2716610: C>T) by real time PCR method. RESULTS In control subjects LPIN1 polymorphism, rs2716610: C>T, was significantly associated with a lower body mass index (BMI) (p=0.008) and waist circumference (p=0.008). The second analyzed rs11693809: C>T polymorphism was associated with lower blood HbA1c levels (p=0.048) in a group of MS patients. CONCLUSION Results of our study suggest that rs2716610: C>T polymorphism of LPIN1 gene could have a protective effect against development of metabolic syndrome, while rs11693809: C>T might affect a glucose control in patients with MS.AIM To investigate whether chronic statin treatment after coronary artery bypass grafting (CABG) protects patients from major cardiac events and provides percutaneous coronary intervention (PCI) free survival. METHODS A total of 232 patients with previous CABG and chronic statin therapy were selected retrospectively and were divided into two groups according to a dosage of atorvastatin per day, e. g., 20 mg or 40 mg. Groups were compared for the major cardiac events and freedom from PCI by Kaplan Meier analysis as the primary end point. Patency of grafts including left internal thoracic artery (LITA) and saphenous vein (SVG) and progression of non-grafted native vessel disease were also evaluated as secondary end points. RESULTS Cardiac mortality, periprocedural myocardial infarction (MI), target vessel revascularization and percutaneous coronary intervention free survival were as follows: 2.9% versus 2.1% (p=1.000); 16.1% versus 21.1% (p=0.331); 56.93% versus 52.63% (p>0.005); 58.4% versus 63.2% (log-rank test; p= 0.347) in atorvastatin 20 mg and atorvastatin 40 mg groups, respectively. However, these results were not statistically significant between two groups (p>0.005). Patency of openness of grafts including LITA and SVG and progression of non-grafted native vessel disease were similar between groups (p=0.112, p=0.779, p=0.379 and p=0.663, respectively). CONCLUSION Low-dose long-term statin treatment had similar outcomes on major cardiac events and identical rate of freedom from percutaneous coronary intervention after coronary artery bypass grafting compared with high-dose long-term statin treatment. It is better to start from low dose statin treatment after surgical interventions.

Effects of the PPARG Gene Polymorphisms on Markers of Obesity and the Metabolic Syndrome in Bosnian Subjects

Summary Background: Peroxisome proliferator-activated receptor gamma (PPARg) is a key transcription factor in adipogene-sis, and also regulates a number of genes associated with lipid storage and insulin sensitivity. Single nucleotide polymorphisms (SNPs) in the PPARG gene have been associated with obesity and diabetes. In this study, we explored the relationship of three PPARG gene variants with the metabolic syndrome (MetS) and related traits in a population from Bosnia and Herzegovina. Methods: Anthropometric and biochemical parameters were measured in 43 patients with MetS and 43 healthy controls. Subjects were genotyped for Pro12Ala (rs1801282) and 1431C>T (rs3856806) SNPs by classic PCR–restriction fragment length polymorphism analysis, and for-681C>G (rs10865710) variant by real-time PCR. Results: The genotype distributions for the three polymorphisms were not significantly different between MetS patients and controls. The Pro12Ala and 1431C>T variants were associated with lower body mass index in the control subjects (p=0.012 and p=0.049, respectively). In this group, the carriers of Pro12Ala had also lower waist circumference compared to the wild-type homozygotes (p=0.045). Conclusions: Results of our preliminary study indicate a beneficial effect of a common Pro12Ala variant on the metabolic phenotype in healthy non-obese subjects.

Optimization of high-resolution melting analysis for simultaneous genotyping of two 11β-hydroxysteroid dehydrogenase type 1 gene polymorphisms.

BACKGROUND Polymorphisms in HSD11B1, the gene encoding 11β-hydroxysteroid dehydrogenase type 1 enzyme, have been associated with obesity, metabolic syndrome, and type 2 diabetes. In this study, we present an optimized high-resolution melting (HRM) method for genotyping two common polymorphisms of the HSD11B1 gene: rs846910: G>A and rs45487298: insA. METHODS One hundred DNA samples from patients with polycystic ovary syndrome and healthy controls were genotyped by HRM. The results were compared with those obtained with classic polymerase chain reaction followed by restriction fragment length polymorphism analysis. RESULTS Various approaches were used during HRM specificity optimization. With the optimized method, genotyping accuracy of 100% was achieved. CONCLUSIONS HRM analysis is a fast, simple, and cost-effective method compared with the alternative genotyping approaches. The work required for optimizing the method (improvement of specificity) is minor compared to the advantages.